Exaggerated responses to inhaled irritant chemicals are a hallmark of human airway diseases such as asthma. While the inflammatory processes and structural remodeling caused by respiratory irritants have received considerable attention, the mechanisms responsible for triggering acute episodes of ­irritant-induced airflow obstruction remain poorly understood. The non-selective cation channel Transient Receptor Potential Ankyrin 1 (TRPA1) has emerged as a promising target for therapeutic intervention in respiratory disorders as result of its unique biology that allows it to serve as the primary neuronal sensor not only for many of the hazardous irritants that cause chronic respiratory disease, but also for multiple inflammatory and oxidant mediators triggered by irritant exposure. Herein, we survey current evidence regarding TRPA1’s role as a sensor of an exceptionally wide array of oxidants, inflammatory mediators and hazardous air pollutants. Further, we review studies demonstrating that pharmacological inhibition or genetic disruption of TRPA1 protects laboratory animals from irritant-­triggered expulsion/dilution reflexes. Lastly, we conclude with a comprehensive overview of patented TRPA1 antagonists and highlight recent progress made toward the goal of developing TRPA1-targeted therapeutics.

The mammalian lung houses a heterogeneous assembly of cell types that works in concert to ensure proper oxygenation of blood and removal of the metabolic waste product CO2. Not surprisingly, inhaled foreign materials and the host’s response to them can impair this vital process. Threats to this exquisite system can be broadly classified into two categories: (1) gradual declines in the lung’s capacity to conduct gas exchange caused by inflammation, oxidative stress and aberrant tissue remodeling, and (2) acute chemical, physical, or other environmental threats to gas exchange that are detected by sensory nerves in the respiratory tract (Figure 10.1), triggering expulsion/dilution reflexes that may include sneeze, cough, bronchospasm, and airway secretions. These two categories are by no means mutually exclusive; by contrast, they are intimately linked, as inflamed and pathologically remodeled airways contain mediators that sensitize neuronal reflex arcs. This continued cycle can progress to the point where expulsion/dilution reflexes including airway fluid secretion and bronchospasm become exaggerated to the extent that they themselves now potentially become acute threats to gas exchange.

Figure 10.1 Sensory innervation of the respiratory tract. Sensory nerves that detect mechanical, thermal, and chemical stimuli in the nasal mucosa are carried by the ethmoidal branch of the trigeminal nerve (cranial nerve V) and synapse in the trigeminal nuclei (trigeminal nucleus principalis, oralis, or caudalis) within the brainstem. Sensory fibers that serve similar functions in the larynx, cartilaginous airways and lung parenchyma are carried within the vagus nerve (cranial nerve X), have cell bodies in either the inferior vagal (nodose) or superior vagal (jugular) ganglia, and synapse within the nucleus tractus solitarius. Inputs conveyed through this network regulate both sensations (e.g., urge to cough and dyspnea, or “air hunger”) and autonomic outflow, which profoundly influences airway secretions and caliber.

One of the great challenges of treating respiratory ailments is finding points to intervene that will disrupt the vicious cycle of insult—pathological response—increased sensitivity to insult. The difficulty of this challenge is underscored by the observation that bronchospasm itself is sufficient to elevate indices of pathological airway remodeling in asthmatic airways [1]. The “pathological response” portion of this cycle is diverse and may feature aberrant remodeling including hyperplasia of resident cells such as airway smooth muscle and cells that produce mucus, high levels of oxidative stress, and nonresolving inflammation that may be driven by the innate and/or adaptive immune system [2]. Advances in fields including biochemistry and molecular biology have enabled rapid and pronounced advances in the identification and measurement of biomarkers associated with disease outcomes, and significant progress has been made in the understanding of this aspect of airway pathophysiology. Despite this impressive body of work, which has led to the discovery and development of multiple experimental therapeutics designed to ameliorate aspects of airway inflammation and/or pathological remodeling, we still do not have satisfactory explanations for what causes the sudden and often violent episodes of intermittent airflow obstruction in asthmatics or others with reactive airways.

Atopy (allergy) is, without question, the single most important risk factor for asthma as there is a clear link and considerable overlap between the two phenomena. In fact, it could be argued that it is precisely the repeated validation and robustness of the link between these two phenomena that has limited investigations into other factors contributing to the asthmatic phenotype. This is not a trivial deficit, as many clinical observations should lead one to believe that allergic airway inflammation is not the sine qua non of asthma. Importantly, asthma and atopy frequently occur independent of one another in humans, and the population-attributable risk of asthma using either skin test sensitivity or total serum IgE as an index of atopy often does not exceed 50% [3]. Furthermore, hazardous air pollutants such as ozone [4] and sulfur dioxide [5] must be considered as agents capable of causing asthma-like symptoms, and the response to these irritants does not categorically involve mast cells [6], an observation consistent with the fact that many asthmatics cite their most frequent triggers as things that do not seem to readily involve allergic reactions [7]. As one might predict, asthmatics exhibit an exaggerated response to inhaled hazardous air pollutants; therefore, environmental and occupational irritants may both overtly trigger and increase the sensitivity of noxious respiratory sensations and respiratory expulsion/dilution reflexes. If in fact this increased irritant sensitivity is an aspect of airway pathophysiology that is separate from but partially overlapping with things such as responses to infectious pathogens and allergens, it stands to reason that airway irritability and the agents that increase airway irritability should be the subject of intense investigation.

A large variety of chemicals cause respiratory irritation of some manner in both experimental animals and human subjects. Whereas the chemicals within this list are heterogeneous with regard to many chemical and physical properties, a considerable number of them share one distinctive property: chemical reactivity. This commonality can be readily observed when reading tables such as the one in Ref. [8], which provides a list of hazardous air pollutants thought to cause and/or exacerbate asthma in humans. Many hazardous irritants on this list are α,β-unsaturated carbonyls such as acrolein that can form adducts with electron-rich (nucleophilic) molecules including cysteines, histidines, and lysines within proteins. In many cases, these Michael addition reactions are irreversible under physiological conditions, creating adducted proteins that change conformation enough to exhibit altered activity or, eventually, cease to function. In addition, reactive molecules may also form adducts with and deplete cellular reductants such as glutathione, causing an oxidant burden in cells. Although modern investigators have developed a sophisticated understanding of the chemical biology of oxidation-reduction reactions, the question of how irritant exposures relate to the fixed and gradual declines in lung function, as opposed to the sudden and potentially life-threatening episodes of airflow obstruction that can occur during a disease exacerbation, remains largely unanswered.

Finally, and perhaps most important from a translational perspective, is the question of whether reactive irritants are broadly and nonspecifically toxic, or whether interrupting downstream biological effectors will ameliorate the damage caused by these hazardous reactive irritants. This question has been investigated in laboratory animals, where invasive experiments can yield mechanistic insight beyond what is readily achievable in humans. As one might expect, inhaled reactive irritants cause oxidative stress in airway cells [9], as well as inflammation and tissue damage [10]. One consistent theme that has emerged, however, is that in mammalian laboratory animal species, capsaicin-sensitive sensory neurons in the airways act as both the primary sensors of inhaled irritants and as the principal effector mechanism that serves to dilute and/or expel the irritant on acute exposure. These experiments are often performed by injecting rodents with high concentrations of capsaicin or resiniferatoxin (RTX), both of which selectively disable sensory neurons containing their receptor by triggering Ca2 + influx sufficient to cause persistent desensitization up to and including destruction of nerve terminals. This method has proven effective for years as a way to disable irritant-sensing nerves in the respiratory tract, as it largely abolishes neuronal responses to a diverse collection of respiratory irritants. Intranasal capsaicin desensitization protocols have also provided marked symptom relief in rhinitis patients, yielding strong evidence that similar mechanisms are also present in humans, at least within the nasal mucosa [11]. Although this technique pinpointed a subpopulation of irritant-sensing nerves as vital components of the response to noxious inhaled materials, it did not have the power to discriminate the molecular sensor(s) responsible for these effects.

In 1997, the seminal work of Caterina et al. [12] identified the ion channel then dubbed “VR1” (vanilloid receptor 1; later renamed transient receptor potential vanilloid 1, or TRPV1) as the receptor for capsaicin. This allowed for detailed mechanistic investigations of the heterologously expressed gene product and its pharmacology. Although these studies confirmed that TRPV1/VR1 was the sole sensor of both capsaicin and RTX, the TRPV1 channel failed to account for the broad irritant-sensing capacity of capsaicin-sensitive nerves, as many chemicals that triggered nocifensive reflexes through capsaicin-sensitive nerves did not appear to activate TRPV1 (see, for example, Ref. [13]). A reasonable conclusion that could be reached from this body of work was that although TRPV1 could serve as a critical gateway to introduce cytotoxic levels of calcium into these sensory nerves, it did not serve as the final common sensor of noxious respiratory irritants. This would mean that at least one other toxin sensor must exist within these nerves.

TRPA1, originally named ANKTM1 due to its unusually large number (14 +) of N-terminal ankyrin repeats, was cloned from lung fibroblasts [14], although its function was not examined until Story et al. [15] reported that it was an ion channel primarily expressed in capsaicin-sensitive sensory neurons that responded to cold temperatures in the noxious range. TRPA1 consists of a putative six-transmembrane (6TM) subunit that assembles as a tetramer to form cation-permeable pores, with a putative pore-forming helix between TM5 and TM6. Further studies of the TRPA1 channel have consistently observed that its expression in naïve animals is predominantly limited to capsaicin-sensitive, TRPV1-expressing nociceptive neurons with cell bodies in the trigeminal, dorsal root, and vagal ganglia [16,17].

Following the characterization of TRPA1 as a nociceptor-enriched ion channel, multiple groups began observing its activation by a diverse array of naturally occurring pungent and algogenic compounds, including isothiocyanates such as allyl isothiocyanate (AITC) from mustard oil, cinnamaldehyde, and bradykinin (BK) (indirectly, via its B2 receptor) [16,18]. Numerous other chemicals that cause noxious sensations in humans were soon revealed to be TRPA1 agonists. This collection of chemicals was surprisingly large and structurally dissimilar. The puzzle of how TRPA1 could serve as such a promiscuous chemosensor was solved in two elegant studies that revealed that chemical reactivity was the property shared by many of these structurally diverse TRPA1 agonists [19,20]. These studies also identified specific cysteine (and, in the case of human TRPA1, a lysine) residues within the cytosolic N-terminus of TRPA1 that, when mutated, selectively inhibited channel gating by reactive stimuli. In aggregate, these early studies showed that TRPA1 was expressed in TRPV1-containing neurons that are the key sensors of chemical nociception and that its unusual ability to convert covalent modification of its intracellular residues into channel gating positioned it as a sensor of an unprecedented range of noxious stimuli. This set of properties made TRPA1 the leading candidate for the missing link between reactive chemicals and capsaicin-sensitive nerve activation.

The generation of mice with genetic deletion of TRPA1 was the next major advance in TRPA1 biology. Two independent groups produced these mice using distinct targeting strategies, with results that were only subtly different. Both groups showed that Trpa1−/− mice demonstrated reduced avoidance or pain behaviors to AITC and reduced hyperalgesia following intraplantar BK injections [21,22]. Intriguingly, Bautista et al. extended these findings by discovering that TRPA1 serves as the primary neuronal sensor of acrolein. This revealed the first mechanism via which a mediator of oxidative tissue damage could directly activate a transducer molecule within a nociceptor. This paper was also the first to generate interest in TRPA1 as a therapeutic target for respiratory disease because inhaled acrolein can cause violent respiratory irritation in humans [23], and it is also a hazardous air pollutant generated by multiple sources including auto emissions and cigarette smoke [24].

Following the excitement generated by the results of Bautista and colleagues, other studies examining TRPA1’s role in respiratory irritant sensing soon followed. Nassenstein et al. [25] used single-cell RT-PCR to demonstrate that TRPA1 mRNA was present in TRPV1-containing vagal neurons that project to the airways of mice. Moreover, they demonstrated that the TRPA1 agonist cinnamaldehyde activated capsaicin-sensitive vagal neurons, caused pulmonary C-fiber discharge, and triggered respiratory irritation reflexes in mice. These experiments provided the first evidence of TRPA1 expression and function in the mammalian respiratory tract, but the authors did not use genetic or pharmacological interventions to confirm the role of TRPA1 in their mouse behavioral studies. Soon after, Bessac et al. employed Trpa1−/− mice to provide the first genetic evidence that TRPA1 was necessary for the sensory neuronal activation and respiratory irritation reflexes triggered by pro-oxidant mediators including HOCl [26]. These findings showed that TRPA1 activation was sufficient to trigger nocifensive respiratory reflexes in mammals and that multiple corrosive agents thought to activate nerves via nonspecific tissue damage act primarily through TRPA1.

As alluded to earlier, multiple irritants trigger respiratory reflexes in laboratory animals via activation of capsaicin-sensitive nerves. Cigarette smoke, a causative factor in numerous cardiopulmonary diseases including COPD, is no exception. Lundberg and Saria [27] initially demonstrated that capsaicin desensitization prevents cigarette smoke-evoked plasma extravasation in rat airways. Andre et al. [28] were able to show that sensory nerve activation and neurogenic inflammatory responses caused by cigarette smoke in mouse and guinea pig are TRPA1-dependent and that α,β-unsaturated aldehydes such as acrolein and crotonaldehyde are the primary constituents of cigarette smoke that trigger these responses. These data proved that acute neuronal responses to cigarette smoke in mice and guinea pigs are mediated by TRPA1 and suggest that further investigations into the role of TRPA1 in chronic cigarette smoking-related illness are merited.

Reactive xenobiotics beyond those found in cigarette smoke can act through TRPA1 to cause nerve activation. Acetaminophen/paracetamol is known to be hepatotoxic at high doses primarily due to the reactive metabolite N-acetyl-p-benzo-quinoneimine (NAPQI), although adverse effects of doses closer to therapeutic levels have not been widely explored. Nassini et al. [29] observed that dosing mice with acetaminophen (15-300 mg/kg) generates detectable levels of glutathione-conjugated NAPQI and that exogenously administered NAPQI causes neurogenic inflammatory responses in rodent lung and skin. Birrell et al. [30] first demonstrated that acrolein depolarizes vagus nerves of mouse, guinea pig, and human. The effect is abolished in tissue from TRPA1 knockout animals or by treatment with the TRPA1 antagonists HC-030031 or AP-18. Acute inhalational exposure to acrolein in rodents results in multiple respiratory reflexes, as well as delayed pulmonary neutrophilia and heightened reflex sensitivity [24,31]. Conscious guinea pigs cough following acrolein exposure, a reflex inhibited by the TRPA1 antagonist HC-030031 [30,32]. Importantly, although human studies such as those conducted by Sim and Pattle [23] would never earn approval from modern IRBs, the fact that their observations of the intensity of irritation caused by inhaled aldehydes in humans correlate strongly with the potency of those aldehydes at recombinant human TRPA1 is striking, as it argues strongly, albeit indirectly, that TRPA1 is the primary human sensor of respiratory irritation caused by toxic aldehydes. Birrell et al. [30] were able to demonstrate translational value of their findings in a more ethically acceptable protocol in human subjects, where they showed that the TRPA1 agonist cinnamaldehyde (3-phenyl-acrolein) causes coughing in both humans and guinea pigs in a similar manner. Others have observed cough in guinea pigs caused by TRPA1 agonists [32,33], although TRPV1 may influence these responses under certain conditions [33].

Cigarette smoke contains many other toxins besides α,β-unsaturated aldehydes such as acrolein and crotonaldehyde, and it stands to reason that many of these toxins may activate TRPA1. Formaldehyde is one such chemical, although it is also an occupational and environmental respiratory hazard. Acute formaldehyde exposure causes ocular and respiratory irritation in humans [23], and sufficiently high exposures may cause chronic respiratory symptoms. In rodents, injections of formalin solutions that contain formaldehyde as the active ingredient have also been used in the study of pain for decades, and TRPA1 gene deletion or pharmacological inhibition yielded full efficacy in these models [34,35]. Based on these results, which also identified TRPA1 as the primary formaldehyde sensor in mouse sensory neurons, we speculated that TRPA1 would also serve as the principal effector of acute respiratory irritation caused by formalin, and this was verified when TRPA1-deficient mice did not respond measurably to formalin exposures that triggered robust respiratory irritation reflexes in wild-type (WT) mice [36]. This pathway may also be relevant in chronic exposure scenarios because the TRPA1 antagonist HC-030031 reduced airway inflammation and hyperresponsiveness in a model where BALB/c mice were exposed to both allergen and formaldehyde [37].

In human subjects, nicotine, the primary addictive principle in tobacco, causes irritation and CNS reflex-mediated secretions when applied to the nasal mucosa [38] and retrosternal pain when inhaled [39]. Talavera et al. demonstrated that nicotine activates TRPA1 channels and that intranasal nicotine instillation causes only minor changes in the “enhanced pause” airflow resistance-related plethysmography value in freely behaving Trpa1−/− mice compared to WTs [40]. The ability of nicotine to cause respiratory irritation is likely multifactorial, as nicotinic receptors are also present on sensory neurons [40–42], and in humans, the retrosternal pain caused by inhaled nicotine is inhibited by the nicotinic receptor antagonist hexamethonium [39]. Thus, TRPA1 may be one of several mechanisms through which nicotine evokes respiratory irritation.

The heavy metal cadmium is in group 12 of the periodic table along with zinc and mercury. The identification of zinc as a TRPA1 agonist drew considerably more attention initially [43,44], although cadmium may arguably have comparable relevance in the respiratory tract due to its presence in tobacco. Indeed, as one might predict, because cadmium and zinc have similar chemistry as reflected by the fact that they belong to the same periodic table group, cadmium and zinc both cause comparable activation of TRPA1 [43], and cadmium causes pain behaviors [45] and airway sensory fiber activation [46] that are predominantly mediated by TRPA1 in acute experimental settings. Future results demonstrating how TRPA1 influences the overall toxicity profile of cadmium and whether mercury, one of the best-characterized toxins, exerts its toxicity in part through TRPA1 activation will be eagerly awaited.

Lipopolysaccharide (LPS) is a constituent of Gram negative bacteria that can trigger immune responses via the toll-like receptor 4 (TLR4) [47]. Recently, Meseguer et al. [48] demonstrated that LPS can activate a subset of mouse sensory neurons in vitro. Strikingly, these responses did not require TLR4, although they were abrogated by TRPA1 gene deletion. LPS activation of TRPA1 is also relevant in vivo, as TRPA1 was vital for the pain behaviors and acute tissue vasodilation/plasma extravasation reactions caused by LPS injections. These studies raise the intriguing possibility that TRPA1 may be a TLR4-independent contributor to the net circulatory dysfunction and organ failure caused by sepsis. Further investigations that will prove or disprove this hypothesis, and determine what role TRPA1 plays in the lung damage caused by this condition, are eagerly awaited.

Ozone is a potent oxidant and an air pollutant that can impair lung function. Occupational exposures during the 1950s first illustrated the effects of ozone on pulmonary function, and studies have since shown that acute ozone inhalation results in noxious respiratory sensations and decrements in lung function, with these responses being at least partially sensitive to local anesthetic inhalation [4]. Ozone can cause rapid, shallow breathing that is blocked by inhibiting vagus nerve conduction [49], although the mechanism(s) by which ozone triggered vagal reflexes remained unclear. Recently, Taylor-Clark and Undem [50] demonstrated that the action of ozone on mouse pulmonary C-fibers is ruthenium red-sensitive and that ozone activation of mouse vagal neurons is mediated by TRPA1

Because AITC is a prototypical TRPA1 agonist, it was conceivable that isocyanates such as those used in the manufacture of polyurethane-containing products might also activate TRPA1. If so, this would be a significant discovery because isocyanate exposure can cause chronic airways disease in exposed workers [51], and this likely occurs via multiple mechanisms that are not readily clear [52]. Prior evidence from laboratory animal studies suggested that toluene diisocyanate (TDI), similar to other hazardous inhaled irritants, could cause respiratory responses such as sneezing and airway smooth muscle contraction that were inhibited by capsaicin desensitization [53,54]. These findings are consistent with the hypothesis that TRPA1 mediates the acute nerve activation and respiratory irritation reflexes caused by isocyanates, and indeed, TDI activates heterologously overexpressed human TRPA1 and causes calcium flux in sensory neurons from WT but not Trpa1−/− mice. Moreover, the respiratory irritation reflexes caused by acute intranasal TDI exposure were also dependent on the presence of TRPA1 [36].

Independently, Bessac et al. [55] investigated the effects of methyl isocyanate, the molecule released during the Bhopal disaster, and came to similar conclusions. Their studies showed that methyl isocyanate activates heterologously overexpressed TRPA1 in excised patch clamp recordings and causes TRPA1-dependent calcium flux in mouse sensory neurons. They also showed that either TRPA1 gene deletion or antagonism with HC-030031 markedly reduced irritation behaviors caused by methyl isocyanate. Taken together, these two studies employed multiple complementary approaches to prove that TRPA1 is the primary target and acute in vivo effector of multiple reactive isocyanates, molecules that have posed considerable occupational hazards in manufacturing workplaces.

Volatile gas anesthetics such as desflurane and isoflurane can provoke effects in humans ranging from coughing to laryngospasm. The reasons for these adverse effects have been unclear. One clue to how volatile gas anesthetics cause airway irritation in humans comes from the observation that sevoflurane does not cause these responses to the same extent that isoflurane and desflurane do [56].

Based on this information, it follows that the mechanism by which gas anesthetics cause airway irritation in humans must be present in healthy individuals and engaged less robustly by sevoflurane than other gas anesthetics. Until the last several years, the only mechanistic evidence that animal studies added to this question was that gas anesthetics activate airway vagal capsaicin-sensitive C fibers and cause respiratory reflexes and that sevoflurane is the least potent anesthetic studied in this regard [57]. Thus, the transducer(s) of gas anesthetic reflex action must be located on airway nociceptive vagal sensory fibers. The search for specific molecular entities that could couple sensing of gas anesthetics to noxious respiratory sensations continued until the breakthrough study by Matta et al. [58], who discovered that TRPA1 is activated by multiple gas anesthetics, but not sevoflurane. Although other mechanisms may influence the respiratory effects of gas anesthetics (including potentiation of the capsaicin sensor TRPV1 [59]), the airway resistance increases caused by desflurane can be prevented by pretreating guinea pigs with the TRPA1 antagonist HC-030031 [60], and neuronal activity in the nucleus tractus solitarius (NTS, the brainstem site where many vagal afferent fibers synapse) triggered by laryngeal exposure to desflurane was inhibited by HC-030031 [61]. Volatile gas anesthetics are not the only class of anesthetic capable of activating TRPA1, as etomidate and propofol activate heterologously expressed hTRPA1 channels and native channels in mouse sensory neurons [58].

Voltage-gated sodium channel-blocking local anesthetics including lidocaine also activate TRPA1 [62], as well as TRPV1 [63,64]. Curiously, acute lidocaine inhalation causes a roughly threefold rightward shift in asthmatic airway reactivity to histamine, even though lidocaine itself reduces airflow in asthmatics [65]. One potential explanation for this apparent paradox that is only speculative at the moment is that lidocaine inhibits histamine reactivity by blocking voltage-gated sodium channels in sensory and/or parasympathetic nerves that may be involved in a CNS reflex-dependent component of the histamine response, whereas the asthmatic airway environment may lead to increased expression and/or gating of TRPA1 and TRPV1 channels on airway sensory nerves such that lidocaine may activate them to an extent sufficient to initiate CNS-dependent parasympathetic reflex airflow obstruction.

Oxidative stress, a condition in which reactive oxygen species (ROS) overwhelm cellular reductant capacity to threaten tissue homeostasis, can be detected in most if not all pathological conditions, including diseases of the respiratory tract. When ROS react with unsaturated fatty acids, they generate lipid peroxidation products such as α,β-unsaturated carbonyls that can covalent modify proteins via Michael addition reactions with thiols. These products of lipid peroxidation include 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal (4-HNE), and 4-oxo-2-nonenal (4-ONE) [66], alkenal lipid peroxidation products that possess chemical reactivity similar to acrolein. Indeed, 4-HNE activates heterologously overexpressed human TRPA1 and causes TRPA1-dependent Ca2 + mobilization in mouse sensory neurons, as well as TRPA1-dependent nocifensive behavior when administered acutely [67].

Unlike the majority of other tissues in the body, the respiratory tract encounters atmospheric oxygen and, as such, the oxidation-reduction balance/redox environment is different [68]. When oxygen tension varies markedly from atmospheric levels, by being either reduced (hypoxia) or elevated (hyperoxia), this redox environment becomes perturbed, triggering oxidative stress. Takahashi et al. [69] demonstrated that both hypoxic and hyperoxic conditions can activate TRPA1 through cysteine oxidation as well as through relief of prolyl hydroxylase-mediated inhibition.

Taylor-Clark et al. [70] also provided insight into how the unique redox environment of the respiratory tract could influence TRPA1 activity when they observed relatively modest mouse pulmonary C-fiber activation and neuropeptide-dependent guinea pig airway constriction in response to 4-HNE, although 4-HNE showed in vitro potency comparable to values obtained by others. Equal concentrations of the more-reactive 4-ONE elicited guinea pig airway constriction and vigorous mouse pulmonary C-fiber activation. The airway constriction caused by 4-ONE was abolished by capsaicin desensitization, blockade of tachykinin neurokinin 1 and 2 receptors, or by preincubating 4-ONE with glutathione to neutralize its reactive capacity. The pulmonary C-fiber activation was abolished by TRPA1 knockout, although TRPV1 also factored into the in vitro activation of mouse neurons caused by 4-ONE. In aggregate, these results demonstrate that lipid peroxidation products such as 4-ONE can activate capsaicin-sensitive sensory nerves in guinea pig airway tissue to elicit axon reflex, neuropeptide-dependent airway constriction. Moreover, these results highlight the possibility that the redox balance of the tissue microenvironment where alkenals such as 4-ONE are generated exerts a profound influence on nerve activation by this and related classes of chemically reactive mediators. Further emphasizing this concept, 9-nitro-oleic acid (9-OA-NO2), a product of the nitration reaction that occurs when reactive nitrogen species covalently modify nucleophiles, causes similar effects as 4-ONE, leading to robust TRPA1 activation and TRPA1-dependent mouse pulmonary C-fiber activation [71]. These findings underscore the role of TRPA1 as a potential integrator of multiple pathways linking oxidative stress and inflammation to noxious sensations and reflex hypersensitivity.

Mitochondria provide the vast majority of energy in healthy cells via oxidative metabolism of acetyl CoA. When this process works efficiently, oxygen is reduced to water; however, when mitochondria are damaged and/or their rate of respiration is markedly altered, ROS including superoxide are generated at levels that can lead to cellular damage. Nesuashvili et al. [72] hypothesized that the mitochondrial complex III inhibitor antimycin A, which generates endogenously-produced ROS, could activate TRPA1, thereby creating the first direct link between dysfunctional mitochondria and TRPA1 activity. Their hypothesis proved correct, as antimycin A activated recombinant TRPA1 and caused mouse pulmonary nociceptor discharge predominantly through TRPA1. Although the specific products involved in this process were not identified, the effects of antimycin A were inhibited by multiple reagents that interfere with ROS production. These experiments provide very strong evidence that antimycin A generates mitochondrial ROS production that leads to TRPA1 activation.

Prostaglandins are lipid mediators generated by the metabolism of arachidonic acid by cyclo-oxygenase (COX) enzymes that are generally pro-inflammatory, as well as pro-algesic due to their ability to sensitize nerves including nociceptors [73]. Prostaglandin E2 (PGE2) is one of the most potent known pro-algesic lipid mediators, and it is produced in high levels by the COX-2 enzyme during inflammatory conditions. Prostaglandin D2 (PGD2) is a related lipid mediator produced by mast cells during conditions such as allergic inflammation [74]. The profound effects of both these mediators on sensory neurons have long been presumed to be entirely through their cognate G protein-coupled receptors (GPCRs), a notion supported by the fact that their acute effects are mediated by GPCRs [75]. Over time, however, PGD2 and PGE2 are nonenzymatically degraded into prostaglandin A2 and 15 deoxyΔ12,14 prostaglandin J2, respectively. Neither of these mediators signals through any known prostaglandin GPCR; however, both molecules contain chemically reactive electron-deficient α,β-unsaturated carbonyls within their cyclopentenone rings. These molecules have numerous biological targets, but when Taylor-Clark et al. [76] revealed TRPA1 as a target of reactive cyclopentenone ring-containing prostaglandins, this provided the first mechanism through which COX products could activate nociceptive sensory neurons in a GPCR-independent manner. Nerve activity triggered by 15 deoxyΔ12,14 prostaglandin J2 is sufficient to generate nocifensive reflexes in vivo, as intraplantar injection causes pain behaviors that are absent in TRPA1 knockout mice [77], and intranasal instillation causes respiratory irritation reflexes (our unpublished observations). Together, these observations raise the intriguing possibility that chronic elevation of COX activity in vivo may raise concentrations of PGD2 and PGE2 to levels sufficient to cause a net accumulation of prostaglandin A2 and 15 deoxyΔ12,14 prostaglandin J2, and that activation of TRPA1 by these molecules may be a major effector mechanism of long-term, slowly reversible sensory neuronal hypersensitivity in disease states.

TRPA1 is also activated by 8-iso-PGA2, [76] an isoprostane produced by ROS oxidation of membrane phospholipids rather than COX activity. Materazzi et al. expanded on this observation by demonstrating that intraplantar injection of 8-iso-PGA2 evoked nocifensive reflexes in WT but not Trpa1−/− mice [78]. In total, these results add to the mounting body of evidence that multiple products of nonenzymatic metabolism of fatty acids directly activate TRPA1 and may contribute to COX inhibitor-refractory noxious sensations in humans.

One of the earliest reports to characterize TRPA1 function [15] reported that the channel (at that point referred to as ANKTM1 due to its high number of ankyrin domains) could be activated when the muscarinic agonist carbachol was applied to cells co-expressing a muscarinic receptor and TRPA1. From this pioneering study, a model has emerged whereby TRPA1 may possibly be gated downstream of any Gq-coupled receptor expressed in the same cell membrane. How widely this occurs in physiological settings remains to be determined, although TRPA1 can be activated in experimental situations by activation of PAR2 [79] or BK’s B2 receptor [18]. The exact mechanism(s) through which this occurs have remained elusive, although it is reasonable to speculate that the intracellular Ca2 + elevation triggered by Gq signaling is one contributing factor because intracellular Ca2 + increases can potentiate channel function (and, at higher concentrations, inhibit it) [80].

BK is a nonapeptide pro-algesic and pro-inflammatory mediator that acts on its B2 (or, in some cases, its inducible B1) receptor to cause nerve activation and sensitization by multiple direct and indirect mechanisms [73]. Importantly, kinins including BK may be important mediators of viral symptoms in humans [81,82], and asthmatics demonstrate greater reactivity to BK than healthy subjects do [83]. Thus, whereas BK regulates the activity of many cellular targets (including ion channels) downstream of its Gq-coupled receptor [84], and it may be one of many mediators that activates or sensitizes airway nerve during inflammatory conditions, interrupting BK signaling in respiratory disease does have potential as a strategy to ameliorate respiratory symptoms. Bandell et al. [16] first demonstrated that TRPA1 is a downstream target of BK when they observed that BK produced outwardly rectifying cationic currents in Chinese Hamster Ovary (CHO) cells dually transfected with both TRPA1 and the BK B2 receptor, but not either one singly. Bautista et al. [22] demonstrated that TRPA1 is a downstream target of BK in native sensory neurons and that this is relevant in vivo when they showed that Ruthenium red, extracellular Ca2 + removal, or TRPA1 knockout markedly reduced BK-induced intracellular Ca2 + elevations in mouse trigeminal neurons and that TRPA knockout also abolished thermal hyperalgesia caused by intraplantar BK. In airway nerve terminals within mouse [85] and guinea pig [86,87] airways, BK causes robust action potential discharge in a manner dependent on the B2 receptor and downstream effectors that include TRPV1 and Ca2 +-activated chloride channels [88]. Consistent with the concept that airway sensory fibers are BK-sensitive in a manner at least analogous to somatosensory fibers, BK also causes coughing, an expulsion/dilution reflex presumably triggered by noxious sensations, in guinea pigs. This coughing is markedly reduced by the TRPA1 antagonist HC-030031 [89]. This effect is most likely at the level of the sensory nerve because HC-030031 inhibited BK-induced sensory neuron Ca2 + flux and vagus nerve depolarization in these studies. Taken together, these data indicate that TRPA1 is involved in BK-induced nociceptor sensitization, hyperalgesia, and cough, and that TRPA1 block may provide a target beyond the B2 receptor that could ameliorate the noxious sensations caused by BK.

Recently, TRPA1 has been identified as a downstream effector of multiple intracellular networks beyond direct signaling by reactive carbonyls and mediators acting via the canonical Gq-coupled pathway. Two cytokines that have been linked to the pathology of atopic dermatitis and asthma, IL-31 and thymic stromal lymphopoietin, activate a subset of mouse sensory neurons through their respective receptors to trigger signals that in turn activate TRPA1 [90,91]. In the case of both of these mediators, direct injection into the skin causes robust scratching in mice, and this behavior is markedly blunted by TRPA1 gene deletion. Although future studies will be necessary to determine what sensations and/or reflexes these mediators cause in the airways and what role TRPA1 could play in those phenomena, these results indicate that TRPA1 is capable of serving as a downstream effector that may amplify signals generated by multiple biochemical pathways initiated by diverse soluble ligands. How extensive—and context-dependent—this list is remains to be determined, however.

TRPA1 block or knockout is anti-inflammatory in models of both allergic [92] and cigarette smoke-induced inflammation [93]. Neurogenic inflammation triggered by TRPA1-dependent release of neuropeptides was presumed to be the mechanism that would explain this phenomenon, although, if true, this could be concerning because multiple molecules that block individual or combinations of the neurokinin receptors that are the predominant effector mechanisms of neurogenic inflammation in animal models have shown little benefit in human trials despite demonstrating pharmacodynamic effects against neuropeptide agonists [94]. However, Nassini et al. [93] demonstrated that TRPA1 immunoreactivity is present in nonneuronal cells within mouse and human airways, and that TRPA1 also plays a pro-inflammatory role in a mouse cigarette smoke exposure model that appears to be independent of neurogenic inflammation. These results expand both the potential pathological role of TRPA1 in human respiratory disease and the possible translational potential of this anti-inflammatory effect.

Although TRPA1 was initially identified due to an observed change in its expression in transformed cells, the role of TRPA1 in cancer is still largely unknown. A recent study [95] has implicated TRPA1 in small cell lung carcinoma (SCLC) cell resistance to serum starvation-induced apoptosis. The same study also demonstrated that siRNA-mediated knockdown of TRPA1 in H146 SCLC cells markedly inhibited their anchorage-independent growth, an in vitro measure of metastatic capability. Heightening the translational potential of these findings, the authors also found TRPA1 message in surgically resected SCLC specimens, but not in non-small cell carcinoma or lung tissue without visible tumor burden. These data suggest that TRPA1 activation may serve as a previously unappreciated mechanism linking cigarette smoke with SCLC metastasis and resistance to chemotherapeutics.

Mice with targeted deletion of the Trpa1 gene product have been used extensively to implicate TRPA1 in numerous pathological processes. These studies have provoked considerable interest in TRPA1 as a therapeutic target for respiratory diseases that may serve the unique function of limiting noxious respiratory sensations caused by acute inflammation and irritant exposures and perhaps even reversing long-term reflex hypersensitivity. What these studies do not provide, however, is insight into the pharmacology of TRPA1 and what properties a channel blocker may need to possess to achieve therapeutic efficacy.

Current in vivo studies of TRPA1 blockers are limited by the number of structural scaffolds currently available. AP18 and HC-030031 (see Table 10.2 and Figure 10.2, respectively) were the first published small molecule blockers of the TRPA1 channel [16,96]. Both molecules display fully efficacious block against native and recombinant TRPA1 channels from multiple species within the low μM range, and studies have shown molecules of both structural templates to be selective over many other molecular targets [97,98]. Obtaining consistent results with these two structurally distinct molecules, particularly when combined with genetic techniques such as antisense oligonucleotides or gene disruption, constitutes a data package that can boost confidence in the target as much as any rodent experiments could be expected to.